White Paper: High Purity Low Endotoxin Arginine: Applications in Biopharmaceutical Processing & Biotherapeutic Stabilization

Arginine, also known as L-arginine (symbol Arg or R), is a basic amino acid widely used in biopharmaceutical processing and stabilization of biotherapeutics.

The structural components of amino acid include an α-amino group, an α-carboxylic acid group, and a side chain in a guanidino group. Amino acids arginine (Arg), lysine (Lys), and histidine (His) have basic side chains at neutral pH.

BIOTHERAPEUTICS FORMULATED & STABILIZED BY ARGININE

As described in Table 1, several biotherapeutics including monoclonal antibodies (mAbs), fusion proteins and recombinant proteins are formulated with arginine or its salts. The most common application of arginine is in formulating high concentration antibody formulations, wherein arginine reduces viscosity of the antibody solution.

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APPLICATIONS OF ARGININE IN BIOPHARMACEUTICALS

Mobile Phase Components in Several Chromatography Processes
Affinity chromatography-based purification is commonly employed in antibody bioprocesses, arginine and several other amino acids find application as a mobile phase component. Several studies investigate the role of arginine as an eluent in affinity chromatography (1-3). Shukla et al., describe a computational approach to understand this role of arginine (4).

Viscosity Reduction
Several studies describe the use of arginine hydrochloride (Arg HCl) and an equimolar arginine-glutamate (Arg-Glu) to reduce the viscosity of high concentration (> 250 mg/mL) antibody solutions (5,6). Viscosities of bovine gamma globulin solution (250 mg/mL) and human gamma globulin solution (292 mg/mL) decreased to below 50 cP in the presence of 1,000 mM Arg HCl (5). Arg-Glu or Arg HCl, reduced the viscosity of a ∼250 mg/mL mAb solution up to 6-fold. With Arg-Glu, the viscosity of the mAb solution was reduced to 30 cP and for a polyclonal sheep IgG solution to 17 cP both at ∼250 mg/mL (6).

Refolding, Protein Solubilization & Protein Aggregation
Arginine is often used as a stabilizer during refolding of proteins and a general inhibitor of aggregation (7-9). Arakawa et al. compare the mechanism of suppression of protein interactions by arginine compared to betaine and guanidine hydrochloride (10). The mechanism by which arginine inhibits aggregation is complicated and in general depends upon several factors including the concentration of arginine, concentration of protein and the type of protein being investigated. Several studies investigate the mechanism by which arginine inhibits aggregation and protein stabilization (11, 12). Equimolar mixture of arginine and glutamic acid is known to significantly enhance protein solubility and stability (13-14). This mixture is also known to increase the storage stability of monoclonal antibodies in solution (15).

Lyophilization
The role of arginine in freeze-drying of protein pharmaceuticals has been recently reviewed (16). Studies also describe the lyophilization of protein formulations with arginine and other lyoprotectant like sucrose and mannitol (17-19). These studies highlight the effect of arginine concentration on cake properties and the general appearance of lyophilized formulations.

QUANTITATION OF TRACE METAL IMPURITIES & ENDOTOXIN IN HIGH PURITY ARGININE

Table 2 (a and b) describe below the measured levels of trace metals in four lots of arginine along with limit of quantitation (in ppb).

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The Endotoxin content in Pfanstiehl’s high purity arginine is controlled at < 0.6 EU/g. Controlling the endotoxin content in high purity arginine is important since the arginine is used in a final drug product. Moreover, careful evaluation and selection of raw materials, intermediates and excipients is necessary to provide an efficient microbial control strategy for manufacture of biotherapeutics.

Known for nearly a century for its first-in-class carbohydrate chemistry expertise, Pfanstiehl has built a reputation for high-quality, cGMP-produced, parenteral-grade excipients (ie, trehalose, sucrose, mannitol, galactose, arginine) at its US headquarters north of Chicago. For more information about Pfanstiehl’s Pure Formulation Solutions, visit www.Pfanstiehl.com. 

REFERENCES
1. Understanding the Role of Arginine and Citrate as Eluents in Affinity Chromatography, Developments in Biotechnology and Bioprocessing, Chapter 5, pp 67–86
2. Improved Column Chromatography Performance Using Arginine, American Laboratory, 2007
3. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog. 2004 Sep-Oct;20(5):1301 8.
4. Understanding the Role of Arginine as an Eluent in Affinity Chromatography via Molecular Computations, J. Phys. Chem. B 2011, 115, 11, 2645-2654.
5. Specific Decrease in Solution Viscosity of Antibodies by Arginine for Therapeutic Formulations. Mol. Pharmaceutics 2014, 11, 6, 1889-1896.
6. Viscosity Reduction of a Concentrated Monoclonal Antibody with Arginine·HCl and Arginine·Glutamate. Ind. Eng. Chem. Res. 2016, 55, 43, 11225-11234.
7. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog. 2004 Sep-Oct;20(5):1301-8.
8. Role of Arginine in the Stabilization of Proteins against Aggregation. Biochemistry 2005, 44, 12, 4919-4925.
9. Aggregation suppression of proteins by arginine during thermal unfolding. Protein Pept Lett. 2006;13(9):921-7.
10. Suppression of protein interactions by arginine: a proposed mechanism of the arginine effects. Biophys Chem. 2007 Apr;127(1-2):1-8. Epub 2006 Dec 21.
11. Review: Why is arginine effective in suppressing aggregation? Protein Pept Lett. 2005 Oct;12(7):613-9.
12. Interaction of Arginine with Proteins and the Mechanism by Which It Inhibits Aggregation. J. Phys. Chem. B 2010, 114, 42, 13426-13438.
13. A Simple Method for Improving Protein Solubility and Long-Term Stability. J. Am. Chem. Soc. 2004, 126, 29, 8933-8939.
14. Understanding the Synergistic Effect of Arginine and Glutamic Acid Mixtures on Protein Solubility. J. Phys. Chem. B 2011, 115, 41, 11831-11839.
15. The effect of arginine glutamate on the stability of monoclonal antibodies in solution. Int J Pharm. 2014 Oct 1; 473(1-2): 126–133.
16. Arginine as an Excipient for Protein Freeze-Drying: A Mini Review. J Pharm Sci. 2018 Apr;107(4):960-967.
17. Freeze drying of L-arginine/sucrose-based protein formulations, part I. J Pharm Sci. 2015 Jul;104(7):2345-58.
18. Freeze-Drying of L-Arginine/Sucrose-Based Protein Formulations, Part 2. J Pharm Sci. 2015 Dec;104(12):4241 4256.
19. Mannitol/l-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals. J Pharm Sci. 2016 Oct;105(10):3123-3135.